ReviewCombinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris

•A CRISPR/Cas9 system has been developed for the methylotrophic yeast P. pastoris.•Several parts for expression (promoters for CAS9 and gRNAs, CAS9 CDSs…) needed optimization.•Only 6/95 constructs designed achieved high efficiency genome targeting.•Targeting rates close to 100% and multiplexing were demonstrated for several genes.•This report may serve as a guideline to implement CRISPR/Cas9 in other eukaryotes.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is one of the most commonly used expression systems for heterologous protein production. However the recombination machinery in P. pastoris is less effective in contrast to Saccharomyces cerevisiae, where efficient homologous recombination naturally facilitates genetic modifications. The lack of simple and efficient methods for gene disruption and specifically integrating cassettes has remained a bottleneck for strain engineering in P. pastoris. Therefore tools and methods for targeted genome modifications are of great interest.Here we report the establishment of CRISPR/Cas9 technologies for P. pastoris and demonstrate targeting efficiencies approaching 100%. However there appeared to be a narrow window of optimal conditions required for efficient CRISPR/Cas9 function for this host. We systematically tested combinations of various codon optimized DNA sequences of CAS9, different gRNA sequences, RNA Polymerase III and RNA Polymerase II promoters in combination with ribozymes for the expression of the gRNAs and RNA Polymerase II promoters for the expression of CAS9. Only 6 out of 95 constructs were functional for efficient genome editing.We used this optimized CRISPR/Cas9 system for gene disruption studies, to introduce multiplexed gene deletions and to test the targeted integration of homologous DNA cassettes. This system allows rapid, marker-less genome engineering in P. pastoris enabling unprecedented strain and metabolic engineering applications.